LPS with reduced toxicity from genetically modified gram negative bacteria

ABSTRACT

The subject invention lies in the field of vaccines. Specifically new compounds that can be used as adjuvants are provided. Recombinant LPS having a reduced number of secondary acyl chains per molecule of LPS vis a vis the corresponding non modified LPS molecule, said secondary acyl chains being bound to primary acyl chains, said primary acyl chains being bound to the glucosamine of said recombinant LPS molecule, said recombinant LPS being homogenous in acylation pattern is an example of such a compound. Also recombinant LPS having a phosphate group attached to the glucosamine at the non reducing end of the LPS molecule and a phosphate group attached to the glucosamine at the reducing end of the molecule per recombinant LPS molecule provides a further example.

This application is a 371 of PCT/NL98/00633 Nov. 3, 1998.

BACKGROUND TO THE INVENTION

The subject invention lies in the field of vaccines and morespecifically provides novel compounds that can be used as adjuvants invaccines. Many adjuvants have been described e.g. Freund type mineraloil emulsions, aluminium salts, saponins, muramyl dipeptide andderivatives MPL, MF59 etc. However only a few have actually beenlicensed for use in humans. This is generally due to an unfavorableratio between immunostimmulatory action versus toxicity. A generalreference concerning adjuvants can be found in The Theory and PracticalApplication of Adjuvants (D.E.S. Stewart-Tull ed. John Wiley & Sons1995) and the information therein is incorporated by reference. Theprior art also teaches for a number of organisms that enzymatictreatment of LPS can lead to reduced toxicity. The LPS illustrated ashaving undergone such treatment are: Salmonella typhimurium andSalmonella minnesota. The following are also suggested to exhibit such:all Gram negative bacteria and specifically Salmonella, Escherichia,Haemophilus, Moraxella, Campylobacter and Neisseria. Nowhere however aredetails provided concerning proof of adjuvant activity.

Looking at this prior art in detail shows that Munford et al (in U.S.Pat. No. 4,929,604 issued in 1990) show S typhimurium LPS in which 95%of secondary acyl groups have been removed through enzymatic treatment.The Munford treatment cannot specifically remove secondary acyl chainsensuring only partial deacylation. The Munford method cannot provideuniform product at best nearly all secondary acyl groups will beremoved.

They suggest adjuvant activity could be present due to B cellmitogenicity testing. B cell mitogenicity testing however is not areliable test to indicate adjuvant activity. It is probable that suchproduct will not exhibit adjuvant activity. The Munford method in factonly shows removal of secondary acyl chains from the non reducing end ofLPS. The resulting product does not contain any secondary acyl group onthe reducing end of the LPS. The Munford product lacks both myristoyland lauroyl secondary side chains. The Munford method cannotspecifically remove only myristoyl or only lauroyl. The Munford methodcannot remove only secondary acyl chain from one specific location. TheMunford method is suggested to also be applicable to Escherichia,Haemophilus and Neisseria.

They show a Salmonella LPS with one phosphate group on the non reducingend and one on the reducing end. The Salmonella LPS has 1 myristoyl and1 lauroyl group on the non reducing end. The Salmonella LPS has nosecondary acyl group on the reducing end.

Myers et al in U.S. Pat. No. 4,912,094 use alkaline hydrolysis undercontrolled conditions to remove only the beta-hydroxymyristic acylresidue that is ester linked to the reducing end glucosamine at position3. Thus a product in which one of the primary acyl chains has beenchemically removed is described. Nothing is mentioned vis a vissecondary acyl chain removal. The resulting product is stated to be lesstoxic and maintains antigenic properties. This is merely stated based onreduced mitogenicity of MPL A (acid hydrolyzed) vis a vis B cellproliferation for the deacylated version. B cell mitogenicity testinghowever is not a reliable test to indicate adjuvant activity.Escherichia coli and Salmonella minnesota LPS are given as examples.Only biological activity data are however given for the Salmonellaminnesota LPS. They suggest the method to be applicable to all LPS butoffer no support thereof.

The same subject matter is discussed in an article of Erwin et al withMunford as co-author (1991). Quoting from the abstract of the Erwinarticle itself the following is remarked in the abstract “These studiesindicate that the contribution of secondary acyl chains to thebioactivities of a given LPS cannot be predicted with confidence fromthe reported structure-activity relationships of Lipid A or from thebehavior of other deacylated LPS.”

Genes involved in lipid A acyloxyacylation are known in the art.Recently two late functioning acyltransferases of lipid A biosynthesisin Escherichia coli were identified as the products of the htrB and msbBgenes (Clementz et al., 1996,1997); the hrtB gene was previouslydescribed as required for growth on rich media above 33° C., and themsbB gene as a multicopy suppressor of htrB. In the optimal reaction,HtrB transfers laurate to (KDO)2-lipid IVA, after which MsbB can addmyristate to complete lipid A acylation (FIG. 1). The predominantproducts formed by htrB and msbB mutants are tetra- and penta-acylspecies, respectively. The genes display 27.5% identity; a third genebelonging to this family is also present in the E. coli chromosome, butits function in lipid A biosynthesis remains to be demonstrated.

The Haemophilus influenzae genome sequence contains both htrB and MsbBhomologues; mutation in htrB is associated with modification of bothphosphorylation and acylation of LPS (Lee et al., 1995), suggesting apleiotropic effect of the loss of the acyloxyacyl chains on decorationof the oligosaccharide chain. A knockout mutation in the H. influenzaehtrB gene was shown to reduce LPS-associated toxicity (Nichols et al.,1997).

Apicella (also author of the cited Lee et al document) et al alsodescribe a htrB knockout mutant in WO97/19688. They described a H.influenzae tetra acyl mutant obtained via a mutation in htrB said mutantLPS supposedly having substantially reduced toxicity yet with retainedantigenicity.

They used homology of E coli htrB sequence to find a similar sequencefor Haemophilus, This similar sequence had 56% identity and 73%similarity to the E. coli htrB sequence. Mutants of H. influenzae weremade and grown. Analysis of the mutant Haemophilus LPS revealedreduction in phosphoethanolamines, 50% less with two in the inner core.A species being a mono or diphosphoryl pentaacyl Lipid A of H.influenzae missing one of the secondary acyl chains (e.g. myrisitic acidmoiety) in about 10% is also revealed by Apicella. In addition atetraacyl was illustrated as having been present in about 90%. Thus theApicella method produces a mixture of recombinant H. influenzae LPSstructures wherein the majority product has no secondary acyl chains.Bactericidal assays of LOS preparations are provided by Apicella as areinfant rat model and chinchilla immunisations using the mutant H.influenzae strain. The tests use LPS per se as immunogen they do notillustrate or suggest anything concerning adjuvant activity. The immuneresponse against LPS per se is exhibited in the tests of Apicella et al.

A Salmonella mutant is also disclosed. This mutant was achievedfollowing the method analogously to the one for H. influenzae TheSalmonella mutant provides an LPS in which the 3′substitution on the Nlinked C14 is a C16 rather than a C12 fatty acid. This embodiment wastenfold less toxic than wild type. No details on antigenicity areprovided for this substance.

They suggested the method could also be applicable to Neisseria,Moraxella, and Campylobacter. In Example 6 e.g. Apicella suggestedanalogous steps to the H. influenzae could be carried out for Neisseriabut nothing is illustrated and the method has clearly not been carriedout. To date no teaching concerning such gene in lipid A synthesis ofNeisseria has been found and no details of tests wherein the geneinvolved in this stage of lipid A synthesis of Neisseria have beenprovided.

The Apicella prior art document reveals that mutation in Salmonellaappears to induce another acyltransferase rather than resulting inomission of secondary acylation in contrast to the result provided forH. influenzae. This illustrates unpredictability in the result whenmutating genes associated with lipid A synthesis in various Gramnegative organisms and is in line also with the teaching of Erwin andMunford.

The Salmonella product is a hepta or hexaacyl i.e. has the same numberof secondary and primary acyl chains as the non mutant. The H.influenzae product is in majority (90%) free of secondary acyl chainsbut also provides a mixture of pentacyl structures. No difference inactivity is provided for any of the various structures or indicated.

The lipid A structure of Neisseria meningitidis had previously beenanalyzed by Kulshin et al in 1992. However nothing is known concerninggenetic make up of Neisseria with regard to presence or absence of ahtrB gene or identity thereof. In addition nothing is known of theinfluence any mutation in such a gene if it could be found would have onthe resulting mutant strain or on the resulting product or products.

SUMMARY OF THE INVENTION

We searched for and identified a genetic sequence involved withsecondary acylation of LPS. We found two different sequences in theNeisseria meningitidis genome. On the basis of this information i.a. wehypothesized the existence of two acyloxyacyl transferases which couldwork in a number of ways. One such manner could be that only one ofthese transferases would catalyze an addition analogous to the processof E. coli, i.e. HtrB (FIG. 1). Alternatively, a single enzyme mightcatalyze both acylations, as the meningococcal lipid A has a symmetricalstructure. We thus undertook mutations in the Lipid A synthesis genes ofNeisseria meningitidis and found that the mutant strains were viable. Wealso found these strains produced mutated LPS. This mutated LPSexhibited reduced toxicity. However mutation in the htrB2 gene resultedin a product that did not retain immunostimulatory activity. It resultedin a product that would not be useful in a vaccine. It resulted in aproduct that could not be used as an adjuvant in a vaccine.

Surprisingly we found however that mutations in the htrB1 gene ofNeisseria meningitidis, did provide a product that was both less toxicand provided adjuvant activity. We analysed the molecular structure ofthe resulting products and arrived at the conclusion that correspondingmolecules from other Gram negative bacteria could be useful in ananalogous manner. We found that not only the toxicity but also theadjuvant activity was closely related to the structure of the molecule.In particular the secondary acyl composition was particularly relevant.We also found the phosphorylation pattern was relevant.

Thus we now provide a method to specifically produce less toxic LPSderivatives with clear adjuvant activity, said derivatives being of anature not previously discernible from the prior art and exhibitingcharacteristics neither known nor predictable from the prior art.

DETAILED DESCRIPTION OF THE INVENTION

The invention is directed at novel less toxic forms of LPS that areobtained through genetically modified Gram negative bacteria. Thesenovel LPS structures exhibit adjuvant activity.

The novel LPS structures are defined as recombinant LPS having a reducednumber of secondary acyl chains per molecule of LPS vis a vis thecorresponding non modified LPS molecule, said secondary acyl chainsbeing bound to primary acyl chains, said primary acyl chains being boundto the glucosamine of said recombinant LPS molecule, said recombinantLPS being homogenous in acylation pattern. In contrast to the prior artwhere chemically modified LPS has been described and the geneticallyengineered H. influenzae LPS according to Apicella the novel LPSaccording to the invention can be obtained such that the homogeneity ofthe acylation pattern, specifically also the secondary acylation patternis guaranteed. Naturally this provides a better basis for addition to avaccine with a view to standardisation but also with a view to analysisof activity of the resulting expression product. Quite specifically asuitable LPS according to the invention is a recombinant LPS moleculehaving a reduced number of secondary acyl chains vis a vis thecorresponding non modified LPS molecule, said secondary acyl chainsbeing bound to primary acyl chains, said primary acyl chains being boundto glucosamine in said recombinant LPS molecule, said recombinant LPSmolecule having at least one secondary acyl chain bound to a primaryacyl chain attached to the glucosamine on the reducing end of saidrecombinant LPS molecule. A recombinant LPS according the invention inany of the embodiments provided can have the same number of primary acylchains as the non modified LPS. A recombinant LPS according to theinvention can have the same composition of primary acyl chains as thenon modified LPS. By way of example a recombinant LPS according to anyof the embodiments of the invention mentioned above has 2 primary acylchains attached to the glucosamine at the reducing end per recombinantLPS molecule. In a suitable embodiment the LPS according to theinvention will have a primary acyl chain present at the 3 position ofthe glucosamine at the reducing end per recombinant LPS molecule. Quitespecifically the lauroyl acylation is the target of amendment of therecombinant LPS vis a vis the non modified LPS. Such a recombinant LPScan have a reduced number of secondary lauroyl chains per recombinantLPS molecule in comparison to the non modified LPS. Suitably arecombinant LPS according to the invention may have a reduced number ofsecondary lauroyl chains attached to the non reducing end of therecombinant LPS molecule per recombinant LPS molecule in comparison tothe non modified LPS. An embodiment according to the invention of any ofthe types described above has been found suitable wherein therecombinant LPS has at least one secondary lauroyl chain attached to aprimary acyl chain at the reducing end of the LPS molecule perrecombinant LPS molecule. By way of example such a recombinant has asecondary lauroyl chain on the primary acyl chain at the 2 position ofthe glucosamine at the reducing end of the LPS molecule per recombinantLPS molecule. In particular a recombinant LPS according to any of thepreceeding embodiments, said recombinant LPS having a secondary acylchain on the primary acyl chain at the 2 position of the glucosamine atthe reducing end of the LPS molecule per recombinant LPS molecule has infact been found to be of interest. Another embodiment of interest is arecombinant LPS molecule according to any of the above definitions ofthe invention has 5 acyl chains in toto per recombinant LPS molecule. Inan alternative suitable embodiment the recombinant LPS according to theprevious embodiments of the invention has a phosphate group attached tothe glucosamine at the non reducing end of the LPS molecule and aphosphate group attached to the glucosamine at the reducing end of themolecule per recombinant LPS molecule. In addition to the above afurther embodiment of the invention consists of a recombinant LPS withone phosphoethanolamine group per recombinant LPS molecule. Also afeasible LPS according to the invention has one phosphoethanolaminegroup per recombinant LPS molecule. A recombinant LPS having a phosphategroup attached to the glucosamine at the non reducing end of the LPSmolecule and a phosphate group attached to the glucosamine at thereducing end of the molecule, the latter phosphate group further beingattached to phosphoetlhaniolaminie at the reducing end of the moleculeper recombinant LPS molecule forms a particularly suitable embodiment.Note that any combination of the described elementd of the various LPSembodiments are also considered to fall within the scope of theinvention. Any Gram negative bacterium can serve as source for arecombinant LPS according to the invention. Specifically in this respecta bacterium selected from the group consisting of the following bacteriaNeisseria, Bordetella, Salmonella and Haemonhilus is considered asuitable source. The Neisseria and Bordetella organisms are particularlydamaging and LPS derived from such bacteria are preferred. Neisseriameningitidis and Neisseria gonorrhoae are two suitable candidates fromthe bacteria belonging to the group of bacteria falling within thedefinition of Neisseria, In the examples we have used LPS derived fromthe Neisseria meningitidis strain H44/76. On the basis of this strain wefound the following LPS structure to be extremely useful.

As stated the recombinant LPS according to the invention exhibitsreduced toxicity. THe reduced toxicity can be deteremined using commonassays for toxicity of which a number are provided in the examples butof which any number of others will be apparent to a person skilled inthe art. A recombinant LPS according to any of the embodiments of theinvention will exhibit reduced toxicity vis a vis the corresponding nonmodified LPS. Another substance against which the reduced toxicity canbe tested is MPL when tested using corresponding assays. A recombinantLPS according to any of the embodiments of the invention exhibitsadjuvant activity. A substance against which the adjuvant activity canbe compared is MPL when tested using corresponding assays. A recombinantLPS according to the invention exhibits adjuvant activity higher thanthat of MPL when tested using corresponding assays. Alternatively theadjuvant activity can be compared to that of Rhodobacter sphaeroides LPSand when tested using corresponding assays the LPS according to theinvention will show higher adjuvant activity. Another way to test theadjuvant activity of a recombinant LPS according to the invention isagainst alkaline hydrolyzed meningococcal LPS. A suitable recombinantLPS according to the invention will exhibit adjuvant activity higherthan that of alkaline hydrolyzed meningococcal LPS when tested usingcorresponding assays. The adjuvant activity can be assessed with anantigen directed against the same bacterial group from which the nonmodified LPS was derived. The adjuvant activity can also be assessedwith an antigen directed against a different organism than one belongingto the bacterial group from which the non modified LPS was derived. Theexamples provide illustration of a test of adjuvant activity. Therecombinant LPS according to the invention can be substantially isolatedand purified using standard methodologies for isolating LPS frombacterial cultures.

The subject invention is not only directed at the LPS per se as definedin any of the aforementioned embodiments of recombinant LPS according tothe invention but also at a composition comprising such recombinant LPS.Such a composition can be a composition for stimulating immune reaction.Quite specifically such a composition can be a vaccine with therecombinant LPS as active component in combination with apharmaceutically acceptable carrier. A composition according to theinvention and specifically a vaccine according to the inventioncomprises the recombinant LPS as adjuvant. The composition is preferablyfor stimulating immune reaction against a Gram negative bacterium. Thecomposition can be used for combating infections caused by organismsother than the organism corresponding to that from which the LPScorresponding to that of the recombinant LPS was derived. However it canquite suitably be used for combating the same type of organism. ANeisseria LPS can be used in a vaccine combating a Neisseria infectionbut also for combating a Bordetella infection. It is also envisaged thata vaccine against measles could comprise a recombinant LPS according tothe invention as an adjuvant. A composition according to the inventioncan be free of other adjuvants. Specifically a composition according tothe invention, preferably a vaccine is free of any of the commonly usedadjuvants of commercial vaccines. Suitably a composition according tothe invention is free of the following adjuvants Freund type mineral oilemulsions, aluminium salts, saponins, muramyl dipeptide and derivativesMPL and MF59. Alternatively a vaccine according to the inventioncomprises the commonly used commercial adjuvants in lower dosages thanis currently in practice for commercial vaccine preparations thusexhibiting lower toxicity than the corresponding vaccine without the LPSaccording to the invention and the normal adjuvant composition andamount. For a composition according to the invention to have imunestimulatory action and to be useful as a vaccine it is preferable thecomposition comprises an antigen in addition to the adjuvant forstimulating immune reaction. Suitably the antigen is specific forobtaining stimulating immune reaction against an organism other than theorganism corresponding to that from which the LPS corresponding to thatof the recombinant LPS was derived. It is also an embodiment that acomposition according to the invention comprises an antigen in additionto the adjuvant for stimulating immune reaction, said antigen beingspecific for obtaining stimulating immune reaction against an organismcorresponding to the group of organisms from which the LPS correspondingto that of the recombinant LPS was derived. By way of example aNeisseria antigen and a recombinant Neisseria LPS according to theinvention. This need not necessarily be the same species but they canbe. So a Neisseria meningitidis recombinant LPS can be present togetherwith a Neisseria meningitidis antigen. However the LPS can also bederived from Neisseria gonorrhoeae or from a Bordetella species.Suitably a composition according to the invention will be in a medicinaldosage form. For example an injectible dosage form. Preferably thecomposition according to the invention will occur in a systemicallyacceptable form. The adjuvant and any additional antigen will be presentin amounts suitable for providing immune stimulatory reaction in a humanor animal. It will be present in a non toxic amount or in a tolerablytoxic amount. It is preferred application of the vaccine does notprovide side effects of a distressing nature. The invention alsocomprises the use of a recombinant LPS according to any of theembodiments of the invention as adjuvant in a composition forstimulating immune reaction specifically in a vaccine formulation. Theinvention also covers a method of treatment for stimulating the immunesystem of a human or animal by administration of a recombinant LPS orcomposition comprising such in any of the embodiments described for acomposition according to the invention in a dosage sufficient to provideimmune stimulation. a person skilled in the art of vaccines will be ableto ascertain on the basis of the subject and/or disease or infection tobe combated what formulations and dosage regimes can be applied.Commonly available antigens and vaccine carriers can be used analogouslyto known vaccines. A buffer solution is a suitable example of a carrier.The method of administration can be by means of any common method forexample parenteral (e.g. intravenous or intramuscular) or oral (e.g.using typhoid bacterial cells to encapsulate the active substance(s))administration.

The subject invention also provides a method for producing a recombinantLPS according to the invention. The method comprises culturing arecombinant Gramnegative bacterium, said recombinant gram negativebacterium comprising a mutation in the lipid A synthesis route at thelevel of addition of secondary acyl chains to the primary acyl chainsattached to the glucosamine of the LPS molecule followed by optionallyisolating and purifying the resultant LPS. Specifically the mutation isa mutation in a gene encoding an enzyme associated with secondary acyladdition. As disclosed above a number of synthesis routes are availablein the art for various Gram negative bacteria. Using the data present inthe prior art in combination with the subject matter disclosed in thesubject document one can arrive at various methods for various sGramnegative organisms to provide a recombinant LPS according to theinvention. Using the sequence data for htrB provided for Neisseriameningitidis strain H44/76 one can arrive at corresponding sequences inother organisms e.g. other Neisseria. Introduction of a mutationeliminating expression of an active htrB1 expression product in suchorganism will ensure production of the desired recombinant LPS. Thelocation and identification of the htrB1 gene is provided in detail inthe examples for Neisseria meningitidis strain H44/76. The sequence datagenerated can be extrapolated to other strains. Using available sequencedata and homology of the probe used in the example or taking anotherprobe on the basis of the whole or partial encoding sequence ofNeisseria meningitidis strain H44/76 htrB1 can lead to indication ofalternative htrB sequences in other organisms. In addition to the abovefor the Neisseria organisms htrB1 has been found to be locateddownstream of the ruvc gene thus any gene sequence encoding htrdownstream of a ruvc sequence is a suitable location for introducing amutation. Any gene sequence exhibiting more than 33% homology to theencoding sequence of FIG. 2 in a Gram negative organism is a potentiallocation for mutation to provide a recombinant LPS according to theinvention. Preferably the degree of homology is even higher e.g. higherthan 50% preferably higher than 60%. The closer the homology is to 100%over a stretch of at least 500 bp and more preferably over the wholelength of the coding sequence the better. Alternatively or incombination one can search for an encoding sequence of the same or closeamino acid sequence and mutate the corresponding sequence such that noactive expression product is produced. Quite suitably the sequence islocated downstream of a ruvc sequence. Preferably the mutation of choiceis a mutation in a gene encoding an enzyme associated with secondaryacyl addition to primary acyl chains at the reducing end of the LPS. Asis apparent from the examples this can suitably be a mutation in a geneencoding an enzyme associated with secondary lauroyl addition. Aspecific suitable mutation is in a gene encoding an enzyme associatedwith secondary acyl chain addition at the primary acyl chain present atthe 2′ position of the glucosamine at the non reducing end of the LPSmolecule.

In an embodiment according to the invention the recombinant LPS isisolated and purified such that it is free of any other forms of LPS. Ina method of formulating a vaccine it is preferred to first isolate theLPS in order for exact formulation of the vaccine to be achieved.Suitably a method according to the invention consists of providing arecombinant LPS having a reduced number of secondary lauroyl chains perrecombinant LPS molecule in comparison to the non modified LPS whichrecombinant LPS is isolated and purified such that it is free of anyother forms of LPS. In a preferred embodiment the recombinant LPS isprovided that has a reduced number of secondary lauroyl chains attachedto the non reducing end of the recombinant LPS molecule per recombinantLPS molecule in comparison to the non modified LPS. In a preferredembodiment the recombinant LPS is provided that has at least onesecondary lauroyl chain attached to a primary acyl chain at the reducingend of the LPS molecule per recombinant LPS molecule. Suitably in such amethod the mutation can be such that the recombinant LPS has a secondaryacyl chain on the primary acyl chain at the 2 position of theglucosamine at the reducing end of the LPS molecule per recombinant LPSmolecule. Suitably the secondary acyl chain is a secondary lauroylchain. A preferred method involves a mutation process resulting in arecombinant LPS according to the invention having 5 acyl chains in totoper recombinant LPS molecule. An alternative method of the inventionconsists of producing a recombinant LPS having a phosphate groupattached to the glucosamine at the non reducing end of the LPS moleculeand a phosphate group attached to the glucosamine at the reducing end ofthe molecule per recombinant LPS molecule. Suitably the LPS product isisolated and purified such that it is free of any other forms of LPS.Alternatively the method can comprise producing a recombinant LPS havingone phosphoethanolamine group per recombinant LPS molecule whichsuitably is isolated and purified such that it is free of any otherforms of LPS.

A method wherein the recombinant LPS having a phosphate group attachedto the glucosamine at the non reducing end of the LPS molecule and aphosphate group attached to the glucosamine at the reducing end of themolecule, the latter further being attached to the phosphoethanolamineat the reducing end of the molecule per recombinant LPS molecule isproduced and preferably is isolated and purified such that it is free ofany other forms of LPS is provided by the invention.

The invention is further illustrated by the examples which are not to beconsidered a restriction on the scope of the invention. The numerousvariants of the LPS and uses thereof according to the invention will beapparent to a person skilled in the art on the basis of informationprovided in the claims, description and figures in combination withcommon general knowledge in the field of genetic engineering,specifically of Gram negative bacteria and vaccine production.Specifically the references cited and information in the DNA databasesaccessible to the public with Gram negative genomic sequences accessibleprior to the filing date are incorporated herein by reference. Wheremethods or processes of isolation, purification are mentioned such arecommon in the art and analogous to other well known procedures. The samecomment is valid for introducing a mutation in the htrB1 gene. This canoccur via insertion, deletion or substitution in a manner known per seonce a DNA sequence of choice to be mutated has been located in anorganism. The method of formulation of a vaccine and administrationthereof are also common procedures that need no further elucidation. Theterms used are art recognised terms for a person skilled in the art thatcan be derived from general text books concerning the field of geneticengineering, Gram negative bacteria and immunology and/or from the citedreferences.

EXAMPLE 1

Construction of Neisseria Meningitidis Mutant htrB 1 with Altered LipidA

Using the htrB/msbB gene sequences from Escherichia coli and Haemophilusinfluenzae, we performed a BLAST search on the gonococcal genomesequences made available on the Internet by the University of Oklahoma.Several contigs with significant homology were identified, and PCRprimers were designed based on these sequences. With meningococcalchromosomal DNA as template, primers pr447-2 and pr670-1 gave a ca. 500bp PCR product which upon cloning in vector pCRII and sequencing wasfound to be homologous to htrB/msbB sequences from several bacterialspecies (33% and 31% respectively for the E. coli genes). This fragmentwas used as probe for isolation of a larger chromosomal fragmentcontaining the complete htrB1 gene of Neisseria meningitidis (FIG. 2).Immediately upstream of this gene an open reading frame with homology tothe ruvC gene from E. coli was found, which presumably is involved inDNA repair and recombination and not LPS biosynthesis.

A kanamycin-resistance cassette was inserted into the BglI site locatedwithin the cloned htrB1 PCR product, and the resulting construct(plasmid pBSNK6, containing also the neisserial uptake sequence) wasused to transform meningococcal strain H44/76 to kanamycin resistance.PCR was used to verify that correct allelic exchange with thechromosomal htrB1 gene had occurred. All transformants thus obtainedshowed an increased mobility of their LPS when analysed byTricine-SDS-PAGE followed by silver staining (FIG. 3).

Binding of monoclonal antibodies specific for the oligosaccharide partof meningococcal LPS was not affected by the mutation, suggesting thatonly the lipid A part must have been altered.

EXAMPLE 2

Structural Analysis of HtrB1 Mutant Lipid A

Fatty acid analysis by gas chromatography/mass spectrometry of wholecells showed a reduced ratio of C12:0/C12:0 3-OH in the htrB1 mutant ascompared to the wildtype parent strain, indicating a (partial) loss ofthe secondary C12:0 acyl chain(s). LPS from this mutant was purifiedthrough hot phenol/water extraction, and the lipid A fraction wasobtained after acid hydrolysis and chloroform/methanol extraction. Itsstructure was subsequently investigated using tandem mass spectrometry.The analysis revealed a major penta-acyl species in which the C12:0acyloxyacyl chain was missing from the nonreducing end of the molecule(FIG. 4).

An additional difference from the parent strain is found in thephosphorylation pattern at the reducing end of the disaccharide, wherean additional phosphate group is present. This mutant lipid A moleculehas a unique structure not found in any of the mutants describedpreviously for other Gram-negative bacteria.

EXAMPLE 3

Biological Activity of HtrB1 Mutant LPS

Whole cells from mutant strain htrB1 were tested for theirLPS-associated biological activity by both the Limulus amebocyte lysate(LAL) and tumor necrosis factor-a) (TNF-a) induction assays. In the LALassay, a 7-fold reduction in activity was observed for whole cells fromthe mutant as compared to the wildtype. For TNF-a induction by MM6cells, htrB1 bacterial cells showed at least a 100-fold reduction inactivity as compared to the wildtype, similar to the reductionpreviously found for whole cells of a completely LPS-deficient mutant(FIG. 5) (L. Steeghs et al 1998). Immunization of mice with outermembrane complexes isolated from the LPS-deficient meningococcal mutantwas used to compare the adjuvant activity of various LPS preparations.Antibody responses were measured in whole-cell ELISA and bactericidalassay against parent strain H44/76.

Immunogenicity of the major outer membrane proteins was restored tonormal levels by both wildtype and htrB1 mutant LPS, but less so by theatoxic LPS species monophosphoryl lipid A (MPL), Rhodobacter sphaeroidesLPS and alkaline-hydrolysed meningococcal LPS (FIG. 6) (Nakano M. andMatsuura M.). Thus, the htrB1 mutant LPS has retained adjuvant activityin spite of decreased toxicity.

EXAMPLE 4

Properties of the HtrB2 Lipid A Mutant

Another gene with homology to htrB/msbB from E. coli and H. influenzaewas similarly identified and inactivated as in the preceeding examplesfor htrB1. In contrast to htrB1 however, LPS from this mutant (termedhtrB2) has strongly reduced adjuvant activity as well as reducedtoxicity (FIG. 6). Also in contrast with htrB1, this mutation could notbe introduced into strain H44/76 with wildtype LPS but only into a galEderivative with a galactose-deficient, truncated oligosaccharide chain.

METHODS

Bacterial Strains and Plasmids

The E. coli strains NM522 and INVaF′ were grown on LB medium at 37° C.The N. meningitidis strain H44/76 and its derivatives were grown at 37°C. on GC medium base (Difco) supplemented with IsoVitaleX (BectonDickinson) in a humid atmosphere containing 5% CO2, or in liquid mediumas described previously (van der Ley et at., 1993). For selection ofmeningococcal transformants (van der Ley et al., 1996) kanamycin wasused in a concentration of 75-100 microgrammes/ml. With E. coli,antibiotics were used in the following concentrations: ampicillin, 100microgrammes/ml; kanamycin, 100 microgrammes/ml. For cloning of PCRfragments, the TA cloning kit with the vector pCRII (Invitrogen) wasused.

Recombinant DNA Techniques

Most recombinant DNA techniques were as described in Sambrook etal.(1989). Plasmid DNA was isolated using the pLASmix kit (Talent). Thepolymerase chain reaction (PCR) was performed on a Perkin Elmer GeneAmpPCR system 9600 with Taq polymerase. Sequence analysis was performedwith an Applied Biosystems automatic sequencer on double-strandedplasmid DNA templates (isolated with Qiagen columns) and with a cyclesequencing protocol.

LPS Analysis

Tricine-sodium dodecyl sulphate polyacrylamide gel electrophoresis wasperformed in 4% stacking and 16% separating gels as described by Lesseet al. (1990). Proteinase K-treated, boiled bacterial cells were used assamples. The gels were run for 17 h at a constant current of 20 mA, andsilver stained by the method of Tsai and Frasch (1982). The chromogenicLAL assay for endotoxin activity was performed using the QCL-1000 kitfrom BioWhittaker Inc. (Walkersville, Md., USA) according to theinstructions of the manufacturer. Overnight cultures were diluted inmeningococcal medium to an OD at 620 nm of 0.1, and serial dilutions ofthese stocks were used as samples in the LAL assay, TNF-a induction bybacterial suspensions was tested with the human macrophage cell-line MM6and quantitated from culture supernatants using the TNF-a sensitive cellline WEHI 164 (Espevik and Nissen, 1986). For fatty acid analysis byGC-MS, OMC samples were acetylated for 3 h at 900C in pyridine andacetic acid anhydride in order to completely dissolve the LPS. Thesamples were subsequently heated for 3 h at 650C in tetrahydrofuran inthe presence of LiAlH4 to reduce the O-linked fatty acids to the freealcohols. These were derivatized to TMS-ethers for 1 h at 600C withBSTFA+1% TMCS in pyridine, and analyzed by GC-MS on an Autospec(Micromass, Man-ches-ter, UK) in the electron impact mode. The amount of3-OH C12 in the samples was quantified using 2-OH C12 as internalstandard. LPS was isolated by the hot phenol-water extraction method(Westphal and Jann, 1965). For isolation of lipid A, LPS was subjectedto mild acid hydrolysis (1% acetic acid, 2.5 h 1000C), followed byprecipitation and final fractionation in chloroform-methanol-water.Structural analysis of purified lipid A was performed with electrospraytandem mass spectrometry. Mass spectrometry was carried out on aquadrupole ion trap instrument (LCQ Finnigan Corp. San Jose USA) fittedwith a nanoelectrospray ion source operated at 600 V. The temperature ofthe inlet capillary was set at 200° C., the maximum number of ions inthe trap at 1,0×10⁷ and the maximum injection time at 150 ms.Nanoelectrospray needles were filled with 2 microlitres of samplesolution. Full MS spectra were recorded over the range 150-1850 amu.Full MS(n) spectra were always preceded by a zoom scan of the parent ionto determine the m/z ratio of the parent more accurately as well astodetermine its charge state. MS/MS spectra were recorded with a windowfor parent ion selection of 3 amu. The excitation energy was adjusteduntil the intensity ratio of the base peak to the parent was between 5and 20. Except for zoom scans spectra were recorded in centroid mode.

Characterization of OMC Composition

Binding of mAbs specific for class 1, 3 and 4 OMPs and for theoligosaccharide part of immunotype L3 LPS was tested in a whole-cellELISA (van der Ley et al., 1995, 1996). Isolation of OMCs by sarkosylextraction and their analysis by SDS-PAGE were done as describedprevious-ly (van der Ley et al., 1993).

Immunization of Mice

Six to eight-weeks old BalB/C mice, five animals each group, wereimmunized on day 0 subcutaneously with 20 microgrammes LPS-deficientH44/76 OMCs supplemented with adjuvant and dissolved in 0.5 ml PBS. Atday 14 and day 28 immunization was repeated and mice were bled at day42. Sera were collected and stored at 4° C. The serum bactericidalactivity against strain H44/76 was assayed as described in Hoogerhout etal. (1995), using a final concentration of 20% rabbit complement. Thebactericidal titer was measured as the reciprocal serum dilution showingmore than 90% killing.

REFERENCES

Clementz, T., Bednarski, J. J. and Raetz, C. R. H.: Function of the htrBhigh temperature requirement gene of Escherichia coli in the acylationof lipid A. J. Biol. Chem. 271 (1996) 12095-12102.

Clementz, T., Zhou, Z. and Raetz, C. R. H.: Function of the Escherichiacoli msbB gene, a multicopy suppressor of htrB knockouts, in theacylation of lipid A. J. Biol. Chem. 272 (1997) 10353-10360.

Espevik, T. and Nissen, M. J.: A highly sensitive cell line, WEHI 164clone 13, for measuring cytotoxic factor/tumor necrosis factor fromhuman monocytes. J. Immunol. Methods 95 (1986) 99-105.

Hoogerhout, P., Donders, E. M. L. M., van Gaans-van den Brink , J. A.M., Kuipers, B.,

Brugghe, H. F., van Unen, L. M. A., Timmernans, H. A. M., ten Hove, G.J., de Jong, A. P. J. M., Peeters, C. C. A. M., Wiertz, E. J. H. J. andPoolman, J. T. Conjugates of synthetic cyclic peptides elicitbactericidal antibodies against a conformational epitope on a class 1outer membrane protein of Neisseria meningitidis. Infect. Immun. 63(1995) 3473-3478. Kulshin, V. A., Z\′e4hringer, U., Lindner, B., FraschC. E., Tsai, C., Dimitriev, A. and Rietschel, E. T.: Structuralcharacterization of the lipid A component of pathogenic Neisseriameningitidis. J. Bacteriol. 174 (1992) 1793-1800.

Lee, N. G., Sunshine, M. G., Engstrom, J. J., Gibson, B. W. andApicella, M. A.: Mutation of the htrB locus of Haemophilus influenzaenontypable strain 2019 is associated with modifications of lipid A andphosphorylation of the lipo-oligosaccharide. J. Biol. Chem. 270 (1995)27151-27159.

Lesse, A. J., Campagnari, A. A., Bittner, W. E. and Apicella, M. A.:Increased resolution of lipopolysaccharides and lipooligosaccharidesutilising tricine-sodium dodecyl sulfate-polyacrylamide gelelectrophoresis. J. Immunol. Meth. 126 (1990) 109-117. van der Ley, P.,van der Biezen, J., Hohenstein, P., Peeters, C. and Poolman, J. T.: Useof tran-sfor-ma-tion to construct antigenic hybrids of the class 1 outermembrane protein in Neisseria meningitidis. Infect.Immun. 61 (1993)4217-4224.

van der Ley, P., van der Biezen, J. and Poolman, J. T.: Construction ofNeisseria meningitidis strains carrying multiple chromosomal copies ofthe porA gene for use in the production of a multivalent outer membranevesicle vaccine. Vaccine 13 (1995) 401-407.

van der Ley, P., Kramer, M., Steeghs, L., Kuipers, B., Andersen, S. R.,Jennings, M. P., Moxon, E. R. and Poolman, J. T.: Identification of alocus involved in meningococcal lipopolysaccharide biosynthesis bydeletion mutagenesis. Mol. Microbiol. 19 (1996) 1117-1125.

Nakano, M. and Matsuura M. in The Theory and Practical Application ofAdjuvants D. E. S. Stewart-Tull ed. John Wiley & Sons 1995 Chapter 14,p315-336.

Nichols, W. A., Raetz, C. R. H., Clementz, T., Smith, A. L., Hanson, J.A., Ketterer, M. R., Sunshine, M. and Apicella, M. A.: htrB ofHaemophilus influenzae: determination of biochemical activity andeffects on virulence and lipooligosaccharide toxicity. J. Endotoxin Res.4 (1997) 163-172.

Sambrook, J., Fritsch, E. F. and Maniatis, T.: Molecular Cloning: ALaboratory Manual. Cold Spring Harbor Laboratory Press, Cold SpringHarbor, New York, 1989.

Steeghs, L., den Hartog, R., den Boer, A., Zomer, B., Roholl, P. and vander Ley P. Nature 392: 449-450 (1998).

Tsai, C. M. and Frasch, C. E.: A sensitive silver stain for detectinglipopo-lysaccharides in polyacrylamide gels. Anal. Biochem. 119 (1982)115-119.

Westphal, O. and Jann, J. K.: Bacterial lipopolysaccharide extractionwith phenol-water and further application of the procedure. MethodsCarbohydr. Chem. 5 (1965) 83-91.

LEGENDS TO THE FIGURES

FIG. 1. Role of the htrB and msbB gene products in Escherichia colilipid A biosynthesis

FIG. 2. Organization (A) and sequence (B) of the htrB1 gene fromNeisseria, meningitidis

FIG. 3. Tricine-SDS-PAGE analysis of LPS from H44/76 wildtype andkanamycin-resistant transformants obtained with plasmid pBSNK6

FIG. 4. Structural analysis by mass spectrometry of lipid A from H44/76wildtype (A) and the htrB1 mutant (B)

FIG. 5. TNF-a induction in MM6 cells by whole bacteria of strain H44/76,mutant htrB1 and LPS-deficient strain pLAK33.

FIG. 6. Comparison of adjuvant activity of various LPS preparations whenused for immunization of mice together with LPS-deficient OMCs.

5 1 1293 DNA Neisseria meningitidis 1 cgggcccccc ctcgaggtca acgtcaatccggcatcgacg ctgatgctcg gtcaggctag 60 gggcgcggca ttggcggcat tggtcagccataagctgccc gtttcggaat acacggcctt 120 gcaggtcaaa caggcggtag tcggcaagggcaaggcggca aaagaacagg tgcagcatat 180 ggtggtgcag atgttgggac tttcgggaacgccccagccg gatgcggcgg acggtcttgc 240 cgtcgcgctg acccacgcct tacgcaaccacgggcttgcc gccaaactca atccttcggg 300 gatgcaggtc aagcgcggca ggtttcaatagtttcagacg gcatttgtat tttgccgtct 360 gaaaagaaaa tgtgtatcga gatgaaatttatattttttg tactgtatgt tttgcagttt 420 ctgccgtttg cgctgctgca caagattgccgacctgacgg gtttgcttgc ctaccttctg 480 gtcaaaccgc gccgccggac cggcgaaatcaatttggcaa aatgtttttc cgaatggagt 540 gaggaaaagc gtaaaaccgt gttgaaacagcatttcaaac acatggcgaa actgatgttg 600 gaatacggtt tatattggta cgcgcctgccggacgtttga aatcgctggt gcgctaccgc 660 aataagcatt atttggacga cgcgctggcggcgggggaaa aagtcatcat cctgtatccg 720 cacttcaccg cgttcgagat ggcggtgtacgcgcttaatc aggatatccc gctgatcagt 780 atgtattccc atcaaaaaaa caagatattggacgaacaga ttttgaaagg ccgcaaccgc 840 tatcacaacg tcttccttat cgggcgcaccgaagggctgc gcgccctcgt caaacagttc 900 cgcaaaagca gcgcgccgtt tctgtatctgcccgatcagg atttcggacg caacgattcg 960 gtttttgtgg attttttcgg tattcagacggcaacgatta ccggattgag ccgcattgcc 1020 gcgcttgcaa atgcaaaagt gatacccgccattcccgtcc gcgaggcaga caatacggtt 1080 acattgcatt tctaccctgc ttggaaatcctttccgggtg aagacgcgaa agccgacgcg 1140 cagcgcatga accgttttat cgaagacagggtgcgcgaac atccggaaca atatttttgg 1200 ctgcacaagc gttttaaaac ccgtccggaaggcagccccg atttttactg actacgtaaa 1260 attacaaaac atatcaggcg tttcagatcaaaa 1293 2 310 PRT Neisseria meningitidis 2 Met Cys Ile Glu Met Lys PheIle Phe Phe Val Leu Tyr Val Leu Gln 1 5 10 15 Phe Leu Pro Phe Ala LeuLeu His Lys Ile Ala Asp Leu Thr Gly Leu 20 25 30 Leu Ala Tyr Leu Leu ValLys Pro Arg Arg Arg Thr Gly Glu Ile Asn 35 40 45 Leu Ala Lys Cys Phe SerGlu Trp Ser Glu Glu Lys Arg Lys Thr Val 50 55 60 Leu Lys Gln His Phe LysHis Met Ala Lys Leu Met Leu Glu Tyr Gly 65 70 75 80 Leu Tyr Trp Tyr AlaPro Ala Gly Arg Leu Lys Ser Leu Val Arg Tyr 85 90 95 Arg Asn Lys His TyrLeu Asp Asp Ala Leu Ala Ala Gly Glu Lys Val 100 105 110 Ile Ile Leu TyrPro His Phe Thr Ala Phe Glu Met Ala Val Tyr Lys 115 120 125 Val Ile IleLeu Tyr Pro His Phe Thr Ala Phe Glu Met Ala Val Tyr 130 135 140 Asn LysIle Leu Asp Glu Gln Ile Leu Lys Gly Arg Asn Arg Tyr His 145 150 155 160Asn Ala Leu Asn Gln Asp Ile Pro Leu Ile Ser Met Tyr Ser His Gln 165 170175 Lys Val Phe Leu Ile Gly Arg Thr Glu Gly Leu Arg Ala Leu Val Lys 180185 190 Gln Phe Arg Lys Ser Ser Ala Pro Phe Leu Tyr Leu Pro Asp Gln Asp195 200 205 Phe Gly Arg Asn Asp Ser Val Phe Val Asp Phe Phe Gly Ile GlnThr 210 215 220 Ala Thr Ile Thr Gly Leu Ser Arg Ile Ala Ala Leu Ala AsnAla Lys 225 230 235 240 Val Ile Pro Ala Ile Pro Val Arg Glu Ala Asp AsnThr Val Thr Leu 245 250 255 His Phe Tyr Pro Ala Trp Lys Ser Phe Pro GlyGlu Asp Ala Lys Ala 260 265 270 Asp Ala Gln Arg Met Asn Arg Phe Ile GluAsp Arg Val Arg Glu His 275 280 285 Pro Glu Gln Tyr Phe Trp Leu His LysArg Phe Lys Thr Arg Pro Glu 290 295 300 Gly Ser Pro Asp Phe Tyr 305 3103 109 PRT Neisseria meningitidis 3 Gly Pro Pro Leu Glu Val Asn Val AsnPro Ala Ser Thr Leu Met Leu 1 5 10 15 Gly Gln Ala Arg Gly Ala Ala LeuAla Ala Leu Val Ser His Lys Leu 20 25 30 Pro Val Ser Glu Tyr Thr Ala LeuGln Val Lys Gln Ala Val Val Gly 35 40 45 Lys Gly Lys Ala Ala Lys Glu GlnVal Gln His Met Val Val Gln Met 50 55 60 Leu Gly Leu Ser Gly Thr Pro GlnPro Asp Ala Ala Asp Gly Leu Ala 65 70 75 80 Val Ala Leu Thr His Ala LeuArg Asn His Gly Leu Ala Ala Lys Leu 85 90 95 Asn Pro Ser Gly Met Gln ValLys Arg Gly Arg Phe Gln 100 105 4 21 DNA Neisseria meningitidis 4atccttcggg gatgcaggtc a 21 5 20 DNA Neisseria meningitidis 5 gaacagattttgaaaggccg 20

What is claimed is:
 1. Lipopolysaccharide (LPS) with a lipid A having asecondary acyl chain only on a primary acyl chain at the reducing end ofthe glucosamine disaccharide.
 2. LPS according to claim 1, wherein thelipid A has a secondary acyl chain on one of the primary acyl chains atthe reducing end of the glucosamine disaccharide.
 3. LPS according toclaim 1, wherein the lipid A has a secondary acyl chain on the primaryacyl chain at the 2 position of the glucosamine at the reducing end ofthe glucosamine disaccharide.
 4. LPS according to claim 1, wherein thesecondary acyl chain is a lauroyl chain.
 5. LPS according to claim 1,with a lipid A having a phosphoethanolamine attached to a phosphategroup at the reducing end.
 6. LPS according to claim 1, with a lipid Ahaving the molecular structure:


7. A composition comprising LPS as defined in claim
 1. 8. A compositionaccording to claim 7 further comprising a pharmaceutically acceptablecarrier.
 9. A composition according to claim 7 comprising an antigen inaddition to the LPS.